A semi-synthetic red light photoswitch for optogenetic control of protein activity

Abstract

Red light is useful for optogenetic control of various protein activities in mammalian deep tissues due to its high tissue penetration, low invasiveness, and low light scattering. However, technology that enables for the optogenetic manipulation using red light remains elusive. Here we develop a red light-activatable, semi-synthetic photoswitch, named MagRed. MagRed is composed of a red light-absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin, and its photo-state-specific de novo synthetic binder. MagRed allows us to reassemble split-proteins using red light and thereby develop a red light-activatable Cre recombinase, which is applicable for mammalian deep tissues. Additionally, we take advantage of MagRed to develop red light-inducible transcription system based on the CRISPR-Cas9 system, enabling for high induction (up to 378-fold) of multiple user-defined endogenous target genes. With high versatility and regulatability, MagRed provides a powerful technology that easily facilitates optogenetics applications for a variety of biological research areas.

Red Light Optogenetics in Neuroscience

“…Homodimerization systems only need one vector to be transported to the target, which decreases the complexity of the study. Heterodimerization systems usually require two vectors, but the small size of certain binding partners may enable the use of a single vector for both components ( Kaberniuk et al, 2021 ; Kuwasaki et al, 2021 ).…”

“…The result, QPAS1, is three times smaller and does not self-oligomerize ( Redchuk et al, 2017 ). However, the QPAS1 system has been shown to have relatively high dark activity, which significantly hinders its future utilization ( Kuwasaki et al, 2021 ).…”

“…Fortunately, two very promising, smaller partners that bind to the photosensory module of a bacterial phytochrome from Deinococcus radiodurans have been published: nanoREDs are optimized nanobodies, that is, engineered single-domain antibodies from camelids ( Huang et al, 2020 ); MagRED is an optimized Affibody, that is, a domain of an immunoglobulin-binding bacterial surface protein ( Kuwasaki et al, 2021 ). Direct comparison of nanoREDs and MagRED has demonstrated their superiority to older QPAS1-based system.…”

“…The D. radiodurans phytochrome has slow dark reversion time, which enables long sustained activation of the optogenetic system (minutes vs. hours). Therefore, their activity is also sustained longer, compared to the QPAS1 system ( Huang et al, 2020 ; Kuwasaki et al, 2021 ).…”

“…(A) Multichromatic directional opsin system BiPOLES ( Vierock et al, 2021 ). (B) Red light-activated CRISPR-Cas 9 system Red-CPTS ( Kuwasaki et al, 2021 ). (C) Red light-activated adenylyl cyclase ( Fomicheva et al, 2019 ).…”